Magnetic nanoparticles

ABSTRACT

A magnetic nanoparticle includes a magnetic core and a superparamagnetic outer shell, in which the outer shell enhances magnetic properties of the nanoparticle. The enhanced magnetic properties of the magnetic nanoparticle allow for highly sensitive detection as well as diminished non-specific aggregation of nanoparticles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/699,378, filed on Nov. 21, 2012, which is a 371 of InternationalApplication No. PCT/US2011/038143, filed May 26, 2011, which claims thebenefit of priority from U.S. Provisional Application Ser. No.61/348,561, filed on May 26, 2010, each of which is incorporated hereinby reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. UO1HL080731-02 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to magnetic nanoparticles.

BACKGROUND

Magnetic nanoparticles that are biocompatible and/or degradable can havebroad applications in biotechnology and medicine. In particular, giventhat biological samples and tissues have intrinsically low magneticsusceptibility, magnetic nanoparticles can offer an efficient contrastmechanism for highly selective detection. For example, magneticnanoparticles can be used in applications including, but not limited to,bio-separation, drug delivery, and gene transfer.

SUMMARY

The present disclosure relates to magnetic nanoparticles and methods fortheir synthesis and use. Each magnetic nanoparticle includes a magneticcore and a biocompatible outer shell, in which the outer shell bothprotects the core from oxidation and enhances magnetic properties of thenanoparticle. The enhanced magnetic properties can include increasedmagnetization and reduced coercivity of the magnetic core, allowing forhighly sensitive detection as well as diminished non-specificaggregation of nanoparticles. By forming biocompatible nanoparticleshaving enhanced magnetic properties, detection of specific targetmolecules, e.g., proteins and single cells, can be improved.

In general, one aspect of the subject matter described in thisspecification can be embodied in a nanoparticle that includes aferromagnetic core and a superparamagnetic shell surrounding themagnetic core.

In some implementations, the nanoparticle has a diameter greater than orequal to about 2 nm. In some cases, the ferromagnetic inner core has acore diameter in the range of about 1 nm to about 15 nm. In someinstances, the super-paramagnetic shell has a thickness greater than orequal to about 0.1 nm. The ferromagnetic core can include Fe, Co, Ni,FePt or SmCo.

In some implementations, the superparamagnetic shell includes an oxideof a magnetic material. In some cases, the super-paramagnetic shellincludes a dopant material. The dopant can include a metal selected fromthe group consisting of Mn, Co, Ni, Zn, and ZnMn.

In some instances, the nanoparticle includes a coating on thesuper-paramagnetic shell, in which the coating is configured to increasethe aqueous solubility of the nanoparticle. The coating can include2,3-dimercaptosuccinic acid (DMSA).

In some cases, the nanoparticle includes a coating on thesuper-paramagnetic shell, in which the coating is configured to bind thenanoparticle to a target molecule.

In some implementations, the nanoparticle includes a dextran polymercoating on the superparamagnetic shell.

In certain implementations, the superparamagnetic shell includes ironoxide.

In another aspect, a method of forming a nanoparticle, the methodincludes forming one or more ferromagnetic nanoparticle cores; andforming a super-paramagnetic shell on each of the one or moreferromagnetic nanoparticle cores. Forming the ferromagnetic nanoparticlecan include combining a metal complex and a surfactant in a firstsolution and annealing the first solution to thermally decompose themetal complex and to form the one or more ferromagnetic nanoparticlecores in the first solution.

In some implementations, forming the super-paramagnetic shell on the oneor more ferromagnetic nanoparticle cores includes, subsequent toannealing the first solution, combining one or more metal-oleatecomplexes to the first solution to form a second solution, and annealingthe second solution to form the super-paramagnetic shell on the one ormore ferromagnetic nanoparticle shells.

In another aspect, a method of determining the presence of a targetmolecule in a subject includes administering to the subject ananoparticle, in which the nanoparticle includes a ferromagnetic core, asuper-paramagnetic shell, and a targeting moiety on thesuper-paramagnetic shell that specifically binds to the targetmolecules, providing sufficient time for the nanoparticle to bind to thetarget molecule, and generating a magnetic resonance image of thesubject, wherein a signal in the image indicates the presence of thetarget molecule.

In another aspect, a method of treatment includes administering to thesubject a nanoparticle, in which the nanoparticle includes aferromagnetic core, a super-paramagnetic shell, and a coating on thesuper-paramagnetic shell to bind to a target cell of the subject,providing sufficient time for the nanoparticle to bind to the targetcell, and applying an alternating electro-magnetic field to the subjectto treat the target cell.

A crosslinked polymer is a polymer in which functional groups on apolymer chain and/or branches have reacted with functional groups onanother polymer to form polymer networks.

A non-crosslinked polymer is a polymer in which few or no individualpolymer chains have reacted with the functional groups of anotherpolymer chain to form an interconnected polymer network.

Magnetic moment is the tendency of a magnet to align with a magneticfield.

Magnetic coercivity is the resistance of a ferromagnetic material tobecoming demagnetized.

Magnetic susceptibility is the degree of magnetization of a material inresponse to an applied magnetic field.

Superparamagnetism refers to materials that do not exhibit magneticproperties when no magnetic field is applied, but behave similar to amagnet upon the application of a magnetic field.

The invention provides several advantages. For example, in someimplementations, the outer shell of the magnetic nanoparticle preventsprogressive oxidation of the nanoparticle core, which, in turn, preventsa reduction in the overall magnetization of the nanoparticle. In somecases, doping the outer shell of the magnetic nanoparticle with metalscan improve the overall magnetization of the nanoparticle. The magneticnanoparticle has the advantage of a stable outer shell, in that theshell's thickness is generally constant over time. In certainimplementations, the outer shell of the magnetic nanoparticle provides abiocompatible surface onto which a target molecule or other bindingmoiety can be attached. Due to the high magnetization of the magneticnanoparticles, they can be useful for highly sensitive detection of suchtarget molecules and/or moieties. Furthermore, due to thesuperparamagnetic shell, the magnetic nanoparticle core exhibits littleor no coercivity at low magnetic fields. Accordingly, non-specificaggregation of multiple magnetic nanoparticles can be avoided in theabsence of an externally applied magnetic field. Conversely, intentionalaggregation of the magnetic nanoparticles can be induced when anexternal magnetic field above a set threshold is applied. Such inducedaggregation can be useful for collection and/or isolation of particulartarget molecules attached to the magnetic nanoparticles.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages will be apparent from the followingdetailed description, the figures and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are cross-sections of exemplary magnetic nanoparticles.

FIG. 2 is a flow chart depicting an exemplary process for fabricating amagnetic nanoparticle.

FIGS. 3A to 3D are images of nanoparticle cores fabricated from metalcomplex:surfactant solutions, each containing a different molar ratio ofthe metal complex to surfactant.

FIGS. 4A to 4F are images of nanoparticle cores fabricated from metalcomplex:surfactant solutions at different reaction temperatures.

FIG. 4G is a graph that shows nanoparticle diameter versus reactiontemperature.

FIG. 5 is a graph that shows normalized magnetization versus temperaturefor Fe@MFe₂O₄ (M=Fe, Co, Mn) magnetic nanoparticles (MNPs).

FIGS. 6a-6b are magnetization curves for Fe@FeO MNPs and Fe@MFe₂O₄(M=Fe, Co, Mn) magnetic nanoparticles.

FIG. 7 is a graph that shows a comparison of transverse relaxivityvalues (r₂) for different magnetic nanoparticles.

FIG. 8 is a schematic representation of a Fe@MnFe₂O₄ magneticnanoparticle before and after coating with dimercapto-succinic acid(DMSA) for conjugation with targeting moieties.

FIG. 9 is a graph that shows normalized ΔT2 versus avidin concentrationfor several types of nanoparticles.

FIG. 10A is a graph that shows relaxation rate per cell concentrationfor several types of MNPs.

FIG. 10B is a graph that shows normalized ΔT2 versus cell concentrationfor Fe@MnFe₂O₄ MNPs.

FIGS. 11A to 11D are MRI images for different magnetic nanoparticles.

DETAILED DESCRIPTION

Preferably, a magnetic nanoparticle has a high magnetic moment toenhance the selectivity of the nanoparticle for detection. Althoughincreasing the nanoparticle size can lead to a higher magnetic moment, ananoparticle that is too large can be difficult to use to detect smallmolecules and cells. Additionally, in some instances, magnetic particlesare susceptible to oxidation, which can reduce magnetization over time.Also, due to the magnetic nature of particles, spontaneous aggregationcan occur, which can make nanoparticles difficult to use in applicationsand which can make selective binding of molecules to nanoparticlesdifficult.

The present disclosure relates to magnetic nanoparticles and methods fortheir synthesis and use. Each magnetic nanoparticle includes a magneticcore and a biocompatible outer shell, in which the outer shell bothprotects the core from oxidation and enhances magnetic properties of thenanoparticle. The enhanced magnetic properties can include increasedmagnetization and reduced coercivity of the magnetic core, allowing forhighly sensitive detection as well as diminished non-specificaggregation of nanoparticles. By forming biocompatible nanoparticleshaving enhanced magnetic properties, detection of specific targetmolecules, e.g., proteins and single cells, can be improved.

Magnetic Nanoparticles

FIG. 1A shows a cross-section of an exemplary magnetic nanoparticle 2.The magnetic nanoparticle 2 includes a nanoparticle core 4 that issurrounded by an outer shell 6. Although shown as having a circularcross-section (a sphere in 3 dimensions), the magnetic nanoparticle 2can have various shapes. For example, the magnetic nanoparticle 2 can beshaped similar to a rod/cylinder, a wire, or a whisker. The nanoparticlecan have other shapes as well. The magnetic nanoparticle 2 also can havevarious sizes. In some cases, the magnetic nanoparticle can have anaverage maximum dimension that ranges anywhere between about 1 nm toabout 20 nm, including, for example, about 2 nm, about 5 nm, about 10nm, about 15 nm, or about 16 nm.

In some implementations, the nanoparticle core 4 is formed fromferromagnetic materials that are crystalline, poly-crystalline, oramorphous in structure. For example, the nanoparticle core 4 can includematerials such as, but not limited to, Fe, Co, Ni, FeOFe₂O₃, NiOFe₂O₃,CuOFe₂O₃, MgOFe₂O₃, MnBi, MnSb, MnOFe₂O₃, Y3Fe₅O₁₂, CrO₂, MnAs, SmCo,FePt, or combinations thereof. The nanoparticle core 4 can have a shapesimilar to the entire magnetic nanoparticle 2. For example, thenanoparticle core 4 can be shaped similar to a sphere, a rod/cylinder, awire or a whisker. The nanoparticle core 4 can have other shapes aswell. The average maximum dimension of the nanoparticle core 4 can varyfrom about 1 nm to about 20 nm including, for example, about 2 nm, about5 nm, about 10 nm, or about 15 nm.

The outer shell 6 of the magnetic nanoparticle 2 partially or entirelysurrounds the nanoparticle core 4. In some implementations, the shell 6is formed from a superparamagnetic material that is crystalline,poly-crystalline, or amorphous in structure. In some cases, the materialused to form the shell is biocompatible, i.e., the shell materialelicits little or no adverse biological/immune response in a givenorganism and/or is non-toxic to cells and organs. Exemplary materialsthat can be used for the shell 6 include, but are not limited to, metaloxides, e.g., ferrite (Fe₃O₄), FeO, Fe2O3, CoFe₂O₄, MnFe₂O₄, NiFe₂O₄,ZnMnFe₂O₄, or combinations thereof. The shell 6 can have a thicknessthat ranges from about 0.5 nm to about 3 nm, including, for example,about 1 nm, about 1.5 nm, about 2 nm, or about 2.5 nm.

The outer shell 6 of the magnetic nanoparticle 2 can serve variousfunctions. For example, when an isolated nanoparticle core without ashell is exposed to an oxidizer (such as oxygen in the air), all or aportion of the core material can oxidize leading to degradation in themagnetic properties of the nanoparticle. In addition, the oxide that isformed can have weak magnetic properties relative to the core material.Forming the outer shell 6 over the core 4, however, can preventundesirable oxidation of the core material and thus preserve themagnetic properties of the core 4. Additionally, in someimplementations, the shell 6 enhances the magnetic properties of thenanoparticle core 4. For example, a magnetic nanoparticle with the shell6 can exhibit a magnetic moment that is about 10 times, about 100 times,or about 1000 times greater than a magnetic moment of a nanoparticlecore formed from the shell material alone.

The magnetization (magnetic moments per unit volume of particles) of themagnetic nanoparticle 2 depends, in part, on the maximum averagedimension of the core 4, the thickness of the shell 6, as well as themagnetization of the core 4 and shell 6, respectively. For example, theoverall magnetization (M₀) of Fe@Fe₃O₄ particles consists of individualcontributions from the Fe core and the ferrite shell, given as given asthe volume-weighted average,

${M_{0} = {{\frac{V_{Fe}}{V_{Fe} + V_{Ferrite}} \cdot M_{Fe}} + {\frac{V_{Fe}}{V_{Fe} + V_{Ferrite}} \cdot M_{Ferrite}}}},$

where M_(Fe) and M_(Ferrite) are the magnetization and V_(Fe) andV_(Ferrite) are the volumes of the core and the shell, respectively.

In some cases, the shell material corresponds to an oxide of the corematerial, in which the oxide is coated over a surface of thenanoparticle core 4 in a controlled process, without further oxidizingthe core 4, rather than being formed from the core material itself. Thiscontrolled shell formation process can produce a shell having a stablethickness over time (i.e., there is little or no increase in the shellthickness) and which has strong magnetic properties. In addition, byforming the shell 6 over a surface of the nanoparticle core 4, there isno reduction in the size of the nanoparticle core.

Due to the superparamagnetic properties of the shell 6, the magneticnanoparticle 2 exhibits little or no magnetic properties when noexternal magnetic field is applied to the nanoparticle 2. In contrast,when an external magnetic field is applied to the nanoparticle 2 above aset threshold, the nanoparticle exhibits the enhanced magnetic momentdescribed above. That is to say, the shell 6 alters the magneticproperties of the nanoparticle 2 such that the entire nanoparticle 2appears to be superparamagnetic. The threshold for exhibiting theenhanced magnetic moment is based on the material and structure of theshell 6.

The reduction and/or lack of a magnetic moment in the absence of anexternally applied magnetic field can, in some implementations, reduceand/or prevent non-specific/spontaneous aggregation of groups of thenanoparticles 2 that can otherwise occur due to their magneticproperties. In contrast, by applying an external magnetic field tomultiple nanoparticles having the core/shell structure described above,it is possible, in some implementations, to induce aggregation of thenanoparticles. Such induced aggregration can be useful when attemptingto detect analytes or other molecules bound to the magneticnanoparticles.

In some implementations, the magnetic moment of the nanoparticle 2 canbe enhanced even further by including a dopant in the shell material.For example, the shell 6 can be doped with metals including, but notlimited to, Mn, Co, Ni, Zn, ZnMn, and combinations thereof. The presenceof dopants in the nanoparticle shell 6 also can affect the point atwhich the nanoparticle 2 exhibits magnetic properties when in thepresence of an externally applied magnetic field. Magnetic nanoparticlecan have the highest magnetic moment when the dopant ion should becomposed level to half of Fe, for example, MnFe2O4, CoFe2O4, NiFe2O4,ZnFe2O4, ZnMnFe2O4.

In some implementations, the magnetic nanoparticle 2 also includes asurface coating. For example, FIG. 1B shows a nanoparticle 2 in which asurface coating 8 is formed on a surface of the superparamagnetic shell6. The surface coating 8 entirely or partially covers the shell 6. Atleast one purpose of the coating 8 is to provide a biocompatible surfacethat can be easily functionalized with targeting moieties, bindinggroups, and the like. In some cases, the surface coating 8 makes themagnetic nanoparticle essentially hydrophilic or hydrophobic. Thesurface coating 8 can be formed of polymers including, but not limitedto, synthetic polymers such as polyethylene glycol or silane, naturalpolymers, derivatives of either synthetic or natural polymers, andcombinations thereof. A natural polymer is obtained when a pure polymer,such as a polysaccharide, is synthesized by a microorganism, plant, oranimal and extracted in substantially pure form. A synthetic polymer isobtained from nonbiological syntheses, by using standard polymerchemistry techniques known to those in the art to combine monomers intopolymers. The polymers can be homopolymers, i.e., synthesized from asingle type of monomer, or co-polymers, i.e., synthesized from two ormore types of monomers. The polymers can be crosslinked ornon-crosslinked Crosslinked polymers are characterized as being heatstable and resistant to breakdown in biological systems. A crosslinkedpolymer has a molecular weight significantly higher than the originalstarting polymer.

In some implementations, the surface coating 8 is not a continuous filmaround the magnetic nanoparticle 2, but is a “mesh” or “cloud” ofextended polymer chains attached to and surrounding the magneticnanoparticle 2. Exemplary polymers include, but are not limited topolysaccharides and derivatives, such as dextran, pullanan,carboxydextran, carboxymethyl dextran, and/or reduced carboxymethyldextran, polymethylmethacrylate polymers and polyvinyl alcohol polymers.In some implementations, these polymer coatings provide a surface towhich targeting moieties and/or binding groups can bind much easier thanto the shell material.

For example, dextran-coated nanoparticles can be made and cross-linkedwith epichlorohydrin. The addition of ammonia reacts with epoxy groupsto generate amine groups. This material is known as cross-linked ironoxide or “CLIO” and when functionalized with amine is referred to asamine-CLIO or NH₂-CLIO. Carboxy-functionalized nanoparticles can beconverted to amino-functionalized magnetic particles by the use ofwater-soluble carbodiimides and diamines such as ethylene diamine orhexane diamine. Avidin or streptavidin can be attached to nanoparticlesfor use with a biotinylated binding moiety, such as an oligonucleotideor polypeptide. Similarly, biotin can be attached to a nanoparticle foruse with an avidin-labeled binding moiety.

In other implementations, the nanoparticles are associated withnon-polymeric functional group compositions. Methods are known tosynthesize stabilized, functionalized nanoparticles without associatedpolymers, which are also within the scope of this invention. Suchmethods are described, for example, in Halbreich et al., Biochimie, 80(5-6):379-90, 1998 and Caroline R. A. Valois et al., Biomaterials,31(2):366-374.

In general, a binding moiety is a molecule, synthetic or natural, thatspecifically binds or otherwise links to, e.g., covalently ornon-covalently binds to or hybridizes with, a target molecule, or withanother binding moiety (or, in certain embodiments, with an aggregationinducing molecule). For example, the binding moiety can be a syntheticoligonucleotide that hybridizes to a specific complementary nucleic acidtarget. The binding moiety can also be an antibody directed toward anantigen or any protein-protein interaction. Also, the binding moiety canbe a polysaccharide that binds to a corresponding target. In certainembodiments, the binding moieties can be designed or selected to serve,when bound to another binding moiety, as substrates for a targetmolecule such as enzyme in solution. Binding moieties include, forexample, oligonucleotide binding moieties, polypeptide binding moieties,antibody binding moieties, and polysaccharide binding moieties.

Synthesis of Magnetic Nanoparticles

FIG. 2 shows a flow chart depicting an exemplary process for fabricatinga magnetic nanoparticle. As shown in FIG. 2, fabrication of the magneticnanoparticle can begin by forming the nanoparticle core (step 200). Tofabricate the nanoparticle core, metal complexes are thermallydecomposed in a solution containing a surfactant. In some cases, thethermal decomposition occurs in an oxygen-free environment (e.g., undervacuum or nitrogen environment) to avoid oxidation of the nanoparticlecores that are formed in the reaction. In an example, a solution of1-octadecene (ODE) and oleylamine (OY) surfactant is heated to atemperature of 250° C. When the temperature of the solution isstabilized, a metal complex, such as Fe(CO)₅, is added to solution andstirred. Fe magnetic nanoparticle (Fe MNP) cores form and precipitateout of the solution. The solution then is cooled to room temperature.Examples of other metal complexes include, but are not limited to,Fe(acac)₂ (where acac is acetylacetonate), Fe(acac)₃, cobalt complexes,nickel complexes, and. Examples of other surfactants include, but arenot limited to, oleyamine, oleic acid and other chemicals containingamine or carboxylic acid chemical functional groups.

The nanoparticle core size depends, in part, on the molar ratio of metalsource and surfactant. In some implementations, decreasing levels ofsurfactant relative to the metal source can result in increasingparticle size. For example, FIG. 3 shows images of Fe nanoparticle coresfabricated from a solution containing OY and an iron metal complex, inwhich the molar ratio ([Fe]:[OY]) was varied at a fixed temperature from5:1 to 35:1. When the ratio changed from 5:1 to 12:1, the growth of theparticle diameter was only about 2 nm. Once the ratio exceeded 12:1, theparticle size stabilized at about 2 nm.

The nanoparticle core size also can depend on the temperature of thereaction during which the nanoparticle cores are formed. In someimplementations, raising a temperature at which the reaction takes placeleads to increasing particle size. For example, FIGS. 4A to 4F showimages of Fe nanoparticle cores fabricated from a solution containing OYand an iron metal complex, in which the temperature of the reactionduring which the nanoparticle cores were formed was varied from 140° C.to 260° C. at a fixed molar ratio ([Fe]:[OY]). As the reactiontemperature was raised stepwise from 140° C. to 260° C., thenanoparticle core diameter increased from about 4.4 nm to about 14.5 nm.

In each of the images of the exemplary nanoparticle cores shown in FIGS.3 and 4, a natural amorphous ferrite shell (FeO) that is formed fromexposure to air and subsequent oxidation of the nanoparticle corematerial is visible. The natural amorphous shell has weak or no magneticproperties and can gradually thicken over time so as to transform the Fecore into the amorphous oxide. The foregoing diameters of the exemplarynanoparticles were estimated by adjusting for the volume of the ferriteshell. FIG. 4G is a graph that shows the nanoparticle core diameter withthe naturally occurring oxide shell and the estimated nanoparticle corediameter without the naturally occurring oxide shell versus reactiontemperature.

Referring again to FIG. 2, following fabrication of the nanoparticlecore, a synthetic shell is formed (step 202) on a surface of the coreparticle. To fabricate the shell, a separate solution containing metalcomplexes can be prepared and then added to the solution containing themagnetic nanoparticle cores. To prevent the natural amorphous ferriteshell from forming prior to fabricating the synthetic shell, thesolution containing the nanoparticle cores can be maintained in anoxygen-free environment (e.g., under vacuum or primarily nitrogenenvironment). The combined mixture then can be annealed to form thenanoparticle shell on a surface of the nanoparticle core.

In an example, a solution of iron-oleate complex is separately preparedfrom the foregoing solution containing the Fe MNP cores. In particular,a solution containing Fe(CO)₅, oleic acid (OA), and ODE is prepared andannealed, preferably under an air-free environment. After cooling, thenew solution then is transferred to a solution containing the Fe MNPcores. The temperature of the mixture containing the Fe MNP cores andmetal complexes then is increased to form a synthetic shell around theFe MNP cores. In particular, the reactor temperature is slowly heated toan optimal annealing temperature (e.g., 300° C.) to form Fe MNPs havinga Fe core and a synthetic polycrystalline ferrite shell (Fe₃O₄), inwhich the synthetic shell exhibits superparamagnetic properties. Thesynthetic shell serves as barrier which prevents oxidation of the coreand is generally stable, exhibiting no change in thickness over time.After cooling the mixture, the magnetic nanoparticles havingsuperparamagnetic shells can be collected, e.g., via centrifugation.

In some implementations, the process of forming the synthetic outershell of the magnetic nanoparticle can include adding additionaldifferent metal complexes to the solution containing the MNP cores. Theadditional different metal complexes can contribute one or more dopantsto the synthetic outer shell of a subsequently formed magneticnanoparticle, in which the dopants can enhance the magnetic properties(e.g., magnetic moment) of the MNPs. One or more combinations ofdifferent metal complexes (e.g., Ni(CO)₄, Co₂(CO)₈) can be added to thesolution to form one or more dopants in the synthetic shell. In anexample, Mn₂(CO)₁₀ is added to the foregoing mixture containing the FeMNP cores and the Fe metal complexes, prior to heating to the mixture.As before, the reactor temperature is slowly heated to an optimalannealing temperature (e.g., 300° C.) to form Fe MNPs having a Fe coreand a synthetic polycrystalline ferrite shell (Fe₂O₄) doped with Mn(Fe@MnFe₂O₄).

In some implementations, the MNPs can be further processed to form asurface coating (step 204) on the synthetic shell, in which the surfacecoating includes functional groups to link the MNP to a binding moiety.As previously explained, the surface coating can include afunctionalized polymeric or non-polymeric coating. In some cases, thecoating can increase or decrease the wetting properties of the MNP. Thepolymeric or non-polymeric coating can provide an exposed functionalgroup for binding to a specific or non-specific moiety. For example, thefunctional group can include an amino, carboxyl, or other reactive groupfor binding to a specific or non-specific moiety. In someimplementations, the moiety can include oligonucleotides that haveterminal amino, sulfhydryl, or phosphate groups, among others, that bindto amino or carboxy groups. Alternatively, a binding moiety can beattached to magnetic nanoparticles via a functionalized polymer that isformed on the synthetic MNP shell. Various non-limiting examples ofmethods for synthesizing functionalized, coated nanoparticles aredescribed below.

In an example, a non-polymeric coating of DMSA can be formed on asurface of the synthetic shell (see, e.g., Albrecht et al., Biochimie,80 (5-6): 379-90, 1998). DMSA is coupled to the synthetic ferrite shell,providing an exposed functional group. In another example, carboxyfunctionalized surface coatings can be synthesized on a MNP according tothe method of Gorman (see WO 00/61191). In this method, reducedcarboxymethyl (CM) dextran is synthesized from commercial dextran. TheCM-dextran and iron salts are mixed together and are then neutralizedwith ammonium hydroxide. The resulting carboxy functionalizednanoparticles can be used for coupling amino functionalizedoligonucleotides.

In another example, carboxy-functionalized MNPs can also be made frompolysaccharide coated MNPs by reaction with bromo or chloroacetic acidin a strong base to attach the carboxyl groups. In addition,carboxy-functionalized particles can be made from amino-functionalizedMNPs by converting the amino groups to carboxy groups using reagentssuch as succinic anhydride or maleic anhydride.

In another example, dextran-coated MNPs can be made and cross-linkedwith epichlorohydrin. The addition of ammonia will react with epoxygroups to generate amine groups (see, e.g., U.S. Patent App. Pub. No.20030124194 and U.S. Patent App. Pub. 20030092029, incorporated hereinby reference, Hogemann, D., et al., “Improvement of MRI probes to allowefficient detection of gene expression,” Bioconjug. Chem. 2000.11(6):941-6, and Josephson et al., “High-efficiency intracellularmagnetic labeling with novel superparamagnetic-Tat peptide conjugates,”Bioconjug. Chem., 1999, 10(2):186-91). This material is known ascross-linked iron oxide or “CLIO” and when functionalized with amine isreferred to as amine-CLIO or NH₂-CLIO.

In another example, carboxy-functionalized MNPs can be converted toamino-functionalized magnetic particles by the use of water-solublecarbodiimides and diamines such as ethylene diamine or hexane diamine.In some implementations, avidin or streptavidin can be attached tonanoparticles for use with a biotinylated binding moiety, such as anoligonucleotide or polypeptide (see, e.g., Shen et al., “Magneticallylabeled secretin retains receptor affinity to pancreas acinar cells,”Bioconjug. Chem., 1996, 7(3):311-6). Similarly, biotin can be attachedto a nanoparticle for use with an avidin-labeled binding moiety.

Characterization of MNPs

The properties of the MNPs can be characterized using varioustechniques. For example, the magnetic properties of the MNPs can beevaluated by examining zero-field-cooled and field-cooled magnetizationcurves. In an example, Fe@MnFe₂O₄ MNPs were fabricated and evaluatedusing an extended Stoner-Wohlfarth magnetization model. The model wasdeveloped to explain the magnetic behavior for an ensemble of identical,single domain nanoparticles that are randomly oriented andnoninteracting with one another. We have further extended this model toreflect the real physical system with Fe@MFe₂O₄ particles as summarizedbelow.

-   -   To accommodate the cubic magnetocrystalline anisotropy nature of        Fe and ferrite, we adopted the framework developed by Joffe and        Heuberger.    -   The influence of thermal activation on magnetization was        included by allowing the thermally-activated reversal of        magnetic moments.    -   We used the size distribution functions obtained by TEM analysis        to account for the size variation in an ensemble of particles.

Using the method, in which it was determined that the synthetic shellfollowed a typical superparamagnetic curve whereas the nanoparticle coreassumed a stable single-domain behavior with non-zero coercivity(H_(c)=350 Oe). The total magnetization of the MNP (i.e., thevolume-weighted average of the magnetization of the core and shell),however, had considerably reduced coercivity (H_(c)=40 Oe) compared tothe coercivity of the nanoparticle core. In the example, thesuperparamagnetic contribution from the synthetic shell saturates tooverwhelm the relatively slow magnetization of the nanoparticle core atlow magnetic fields. In contrast, the contribution of the core towardsmagnetization becomes dominant at higher magnetic fields to increase theoverall magnetization of the MNP. Other techniques, such asmicromagnetic simulation and measurements of nuclear magnetic relaxationdispersion, can be used to further characterize the MNPs.

Uses of MNPs

Magnetic nanoparticles can be used in various applications, including inmedicine as biologically compatible and environmentally sensitivesensors and/or molecular imaging agents. For example, the MNPs can beused as magnetic resonance-based sensors in which the MNPs are used asremote sensors for detecting various analytes in an aqueous, (i.e.,water-containing) sample and can be used for the continuous monitoringof changing levels of analytes in the aqueous sample. The MNPs can besuspended or suspendable in an aqueous liquid phase and be covalently ornon covalently linked to, or otherwise have immobilized thereon, one ormore moieties selected to alter the state of aggregation of the MNPs asa function of the presence or concentration of the analyte in thesolution (see, e.g., U.S. Patent App. Pub. No. 20060269965, incorporatedherein by reference).

In another example, the MNPs can be used in aggregate formation assaysto detect target molecules (see, e.g., U.S. Patent App. Pub. No.20030092029, incorporated herein by reference). In the aggregateformation assays, a population of conjugates (or a mixture of two ormore populations of conjugates with differing binding moieties directedto a target molecule or type of target molecule) is placed into a samplesolution. Each conjugate comprises one or more binding moieties (e.g.,an oligonucleotide, nucleic acid, polypeptide, or polysaccharide)linked, e.g., covalently or non-covalently, to a magnetic, e.g.,superparamagnetic, nanoparticle. The binding moiety causes a specificinteraction with a target molecule (or, in some embodiments, anaggregation inducing molecule, such as avidin). The binding moietyspecifically binds to a selected target molecule, which can be, forexample, a nucleic acid, polypeptide, or polysaccharide. As a result,the dispersed state of the conjugates switches to an aggregated state,which decreases a spin-spin relaxation time (T2) of adjacent waterprotons in the aqueous solution. In some cases, the MNPs can be used formagnetic separation of target molecules, e.g., protein.

In another example, the MNPs can be used to detect target molecules inaggregate dispersion assays (see, e.g., U.S. Patent App. Pub. No.20030092029, incorporated herein by reference). In aggregate dispersionassays, conjugates are used to prepare small aggregates, and theaggregates are placed into a sample solution. In this assay system, thebinding moieties are designed so that they can be bound to each other(or to a specific aggregation inducing molecule, such as avidin) to formthe aggregates, and to be (or form upon binding to each other or to theaggregation inducing molecule) a substrate that is cleaved by a specifictarget molecule. If the sample solution contains a target molecule, thesubstrate formed by the binding moieties is cleaved, resulting in thedissolution of the aggregates. Thus, the aggregated state switches to adispersed state, which increases T2 relaxation times.

The aggregates in these assay systems can be observed and detected invitro, e.g., in vials or arrays, e.g., 2-D or 3-D arrays, as well as invivo, e.g., using MR imaging of a subject after administration of theconjugates or aggregates. In some cases, the MNPs can be used forimaging without requiring aggregation of multiple nanoparticles. Furtherexplanation of the foregoing uses can be found in, e.g., U.S. Pat. Nos.6,818,199, 6,203,778, and 5,766,572, Jae-Hyun Lee et al., NatureMedicine 13:95-99; Daniel L. J. Thorek Annals of Biomedical Engineering2006, 23-38.

In another example, the MNPs can be bound to target cells, such ascancer cells, for use in magnetic hyperthermia. In particular,conjugated MNPs can be provided to a cancerous cell or tissue, either invitro or in vivo, in which the conjugated MNPs bind to the cancerouscell/tissue. In some implementations, the MNPs include targetingmoieties that cause the MNPs to bind to specific cancer cells and/orcause the MNPs to travel to specific parts of a subject's body wheninjected systemically. See, e.g., Maite Lewin et al., NatureBiotechnology 18:410-414; PCT No. PCT/KR07/00961. Subsequently, anexternally applied alternating magnetic field (e.g., 100 kHz) is appliedto the bound MNPs such that the motion of the MNPs in response to theapplied field generates an increase in thermal energy to treat thetarget cells/tissue. In particular, the increase in thermal energy canlead to destruction of the cancerous cells/tissue.

In another example, the MNPs can be used in magnetofection. Inmagnetofection, conjugated MNPs are bound to a target molecule, such asnucleic acid, and a magnetic field then is applied to the molecule boundMNPs to deliberately introduce and concentrate the particles into one ormore target cells. The nucleic acids can then be released into the cellcytoplasm by various different mechanisms such as, for example: 1) theproton sponge effect, which is caused by cationic polymers coated on theMNPs that promote endosome osmotic swelling, disruption of the endosomemembrane and intracellular release of the nucleic acid; or 2) thedestabilization of the endosome by cationic lipids coated on the MNPsthat release the nucleic acid into the cell by flip-flop of cellnegative lipids and charge neutralization. See, e.g., U.S. Pat. No.7,635,734 and Eric M Pridgen et al., Nanomedicine 2(5):669-680.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Synthesis and Characterization of Ferromagnetic IronCore/Superparamagnetic Iron Oxide Shell Nanoparticles

We initially prepared Fe MNP cores. 20 mL ODE and 0.3 mL OY (0.64 mmol)were added into a 250 mL 3-neck glass round-bottom flask. A condenserand a thermocouple were connected to separate necks of the flask. Themixture was heated up to 60° C. under vacuum for 1 hour and rechargedwith N₂ gas to completely remove O₂. This procedure was repeated twice.The mixture was then heated to the desired temperature (e.g., 260° C.for 16-nm Fe MNP cores). When the temperature became stable, Fe(CO)₅(1.4 mL, 10 mmol) was carefully injected into the reactor whilstvigorously stirring; the solution quickly turned black as the carbonyldecomposed and nanoparticles began forming. The solution was kept atthis elevated temperature and under N₂ flow for 1 hour, after which itwas cooled to room temperature.

While the Fe-only MNP cores were formed, a manganese and iron-oleatecomplex was separately prepared. Mn₂(CO)₁₀ (156 mg, 0.8 mmol), OA (2.3mL, 7.26 mmol) and 10 mL ODE were added to a 100 mL 3-neck round-bottomflask. The temperature was increased to 60° C. under vacuum for 1 hourand under N₂ flow, which was then repeated twice more. The oxygen-freesolution was heated to 120° C. and Fe(CO)₅ (0.21 mL, 1.61 mmol) wassubsequently injected. The mixture was stirred for 30 minutes. Thesolution turned yellow during this process. No particles, however, wereformed due to the relatively low temperature (120° C.). The solution,containing metal-oleate complexes, was cooled to room temperature andthen carefully transferred to the Fe-only MNP core solution usingdouble-ended needles. The mixture of Fe-only MNP cores and metal-oleatecomplexes was stirred for 30 minutes at room temperature. The reactortemperature was then slowly increased at 5° C./minute. We monitored thecrystallinity of the nanoparticles during the rise in temperature anddetermined the optimal annealing temperature (300° C.) for theferrite-shell formation. When the temperature stabilized, the mixturewas stirred for a further hour. Following completion of the reaction,the solution was cooled to room temperature and 150 mL isopropanolsolution (ODE/isopropanol=0.2 v/v) was added. The Fe@MnFe₂O₄ MNPs werecollected via centrifugation (3,000 rpm, 15 minutes) and dispersed in 10mL hexane. To wash the particles, 50 mL ethanol was added to theparticle solution, the mixture was centrifuged, and the precipitate wasredispersed in 10 mL hexane. These washing steps were repeated threetimes to ensure removal of excess chemicals.

Example 2 Magnetic Properties of Core/Shell Nanoparticles

The size of the prepared nanoparticles was characterized using dynamiclight scattering (Zetasizer Nano-ZS, Malvern). The shape, structure, andcomposition were further characterized using a transmission electronmicroscope (TEM; JEOL 2100, JOEL USA), an X-ray powder diffractometer(XRD; RU300, Rigaku), and an inductively-coupled plasma atomic emissionspectrometer (ICP-AES; Activa-S, HORIBA Jobin Yvon), respectively. Themagnetic properties were analyzed using a vibrating sample magnetometer(EV-5, ADE Magnetics) and a superconducting quantum interference device(SQUID) magnetometer (MPMS-5, Quantum Design).

We characterized the magnetic properties of Fe@MFe₂O₄ MNPs. Forcomparative analysis, we also synthesized ferrite MNPs with differentsizes and compositions. The Ms of all types of Fe-core particles (Fe@FeOand Fe@MFe₂O₄, M=Fe, Co, Mn) was larger than that of ferrite MNPs withFe@MnFe₂O₄ assuming the largest Ms. The ferrite shells had reducedmagnetization compared to bulk material, presumably because of theirpolycrystalline nature. FIG. 5 is a graph that shows zero-field-cooledand field-cooled normalized magnetization for Fe@MFe₂O₄ (M=Fe, Co, Mn)MNPs, in which peaks are displayed at two different temperatures(TB1>TB2). The two peaks denote the separate onset of superparamagnetismin the Fe core (TB1) and the ferrite shell (TB2). The Fe-core hadTB1≈290° C., indicating the stable, ferromagnetic nature of the core atroom temperature.

FIGS. 6a-6b are magnetization curves for Fe@FeO MNPs and Fe@MFe2O4(M=Fe, Co, Mn) MNPs. The magnetization curves were obtained using avibrating sample magnetometer at T=300 K. All measurements wereperformed with MNPs in a powder form and 2 hours after synthesis. Thenumber of MNPs in a sample was quantified using an inductively-coupledplasma atomic emission spectroscope (ICPAES). Initially, we analyzed themagnetic properties of Fe@FeO MNPs (FIG. 6a ). Hysteresis curvesconfirmed the superparamagnetic nature of particles with Fe corediameters up to 11 nm. Overall, Ms was observed to increase more inlarger particles in parallel to the larger Fe core portions. The Ms ofthe FeO shell, which was measured using the shell-only MNPs, wasrelatively small (8 emu/g [Fe]). After compensating for the contributionof the shell, the estimated Ms of the Fe core was 206 emu/g [Fe] (for an11 nm core), which was close to that of the bulk material (210 emu/g[Fe]).

The magnetization curves of Fe@MFe₂O₄ (M=Fe, Co, Mn) MNPs each displayedan unusual feature (FIG. 6b ) of hysteresis loss concurrent withnegligible remanence. In MNPs with the same Fe core size, overallmagnetizations followed the same Ms order as that of the shell material(MnFe₂O₄>Fe₃O₄>CoFe₂O₄). This trend thus serves to validate our approachfor increasing the overall particle Ms via metal doping of the ferriteshell. Table 1 lists the overall Ms of different types of MNPs, the Msof the oxide shell, as well as the contribution of the shell towards theoverall Ms. As the ferrite only MNPs have no core/shell structure, thereis no difference between the overall Ms and the Ms of the iron oxide forthose nanoparticles. The Ms for each MNP type was measured at atemperature of 300 K and an external magnetic field strength ofH_(ext)=10 kOe. The overall diameter of the nanoparticles measured wasabout 16 nm. The estimated Ms of the ferrite shell is smaller to thoseof ferrite only MNPs, possibly because of the multi-domain nature of theshell.

TABLE 1 Overall M_(s) of Properties of iron oxide MNPs M_(s) (emu/gContribution to MNP type^(‡) (emu/g [metal]) [metal]) Overall M_(s) (%)Fe@FeO 92 8 4.9 Fe@Fe₃O₄ 142 58 16.4 Fe Fe@COFe₂O₄ 133 41 12.3 CoreFe@MnFe₂O₄ 149 69 18.8 — — — — Fe₃O₄ 95 95 100 Ferrite CoFe₂O₄ 92 92 100MnFe₂O₄ 101 101 100

For further analysis, the magnetic properties of Fe@MFe₂O₄ MNPs werecalculated using an extended Stoner-Wohlfarth magnetization model, whichincludes the effects of non-zero temperature and cubicmagnetocrystalline anisotropy. According to the simulated results, theshell (M_(shell)) magnetization followed a typical superparamagneticcurve, whereas the core portion (M_(core)) assumed stable single-domainbehavior with non-zero coercivity (Hc=350 Oe). The total magnetizationof a particle (M_(tot)), the volume-weighted average of Mcore andMshell, however, had considerably reduced coercivity (Hc=40 Oe): thesuperparamagnetic contribution (from the M_(shell)) rapidly saturates tooverwhelm the relatively slow magnetization of the core at low magneticfields. The core contribution (M_(core)) then becomes dominant at highermagnetic fields to increase the overall M_(tot).

Example 3 Numerical Simulation of Magnetization

Micromagnetic simulation of Fe@MnFe₂O₄ further corroborated thepostulated magnetization mechanism. In the absence of an externalmagnetic field (H_(ext)) and at T=300 K, the magnetization vectors inthe Fe-core formed a vortex state, which indicated strong exchangecoupling to withstand thermal reversal of the magnetization. In theparticle shell, the vectors were randomly oriented, demonstrating asuperparamagnetic nature. These configurations qualitatively agreed withzero-field-cooled and field-cooled magnetization measurements, whichshowed high TB1 for the Fe-core and much lower TB2 for the shell at roomtemperature. At non-zero field strength, the alignment of magneticmoments was path-dependent and led to the hysteresis loop. A similarsimulation with ferrite MNPs showed no hysteresis and the magnetizationvectors were randomized by thermal energy at H_(ext)=0.

We subsequently characterized the transverse relaxivity (r₂) of theFe-core MNPs. To cross-examine the effects of the Ms and the size (d) ofMNPs on r₂, we also prepared ferrite (Fe₃O₄) MNPs of differentdiameters. FIG. 7 is a comparison of the r₂ values for different MNPs,measured at Larmor frequency f0 (=ω0/2π)=20 MHz and T=300 K. Amongferrite MNPs, the r₂ rose with increased particle size (d). For a fixedparticle size (d=16 nm), the r₂ values were proportional to Ms andcorrespondingly, Fe-core MNPs had higher values of r₂ than ferrite-basedparticles. Fe@MnFe₂O₄ MNPs assumed the highest r₂ due to its highest Ms;the r₂-enhancement over Fe₃O₄ or cross-linked iron oxide (CLIO)nanoparticles was greater than 2-fold and greater than 300-fold,respectively. We further analyzed the r₂ behavior of MNPS using anouter-sphere model that takes into account the water diffusion in r₂calculation. In short, with sufficiently small particle size, thediffusional motion of water molecules is fast enough to average out themagnetic fields from MNPs, and the r₂ in this regime is ˜d²·Ms. Indeed,the observed r₂ values were highly compatible with theoretical values,which 1) validated the approach for enhancing r₂, by increasing particlesize and the magnetization; and 2) verified that the MNPs werewell-dispersed without aggregation to be in the motional-averagingregime (d less than 23 nm for Fe@MnFe₂O₄.

To understand the unusual magnetic properties of Fe@MFe₂O₄ MNPs, weperformed two different levels of magnetic simulations:

-   -   Hysteresis loops were calculated for an ensemble of MNPs, which        included exploring the effects of thermally-activated reversal        of magnetization.    -   The configuration of magnetization vectors in a single particle        was analyzed through micromagnetic simulations.

The results revealed a novel mechanism of magnetization within Fe@MFe₂O₄MNPs. In this mechanism, the Fe core (diameter=12 nm) exhibitssingle-domain, ferromagnetic behavior with appreciable coercivity, whilethe ferrite shell has a superparamagnetic nature and is easilymagnetized at low external magnetic fields (Hext). When these twocomponents (core and shell) are combined together, the coercivity isreduced as the shell leads the initial magnetization at low Hext, butthe hysteresis loss associated with the core is retained at higher Hext.The proposed model consequently explains the magnetic behavior observedin Fe@MFe₂O₄ MNPs in temperature and external field dependentmagnetization.

Example 4 Surface Modification and Conjugation with Biotin or Antibody

Fe@MnFe₂O₄ MNPs were suspended in 10 mL chloroform, and 50 μLtriethylamine was added. DMSA (50 mg) in 10 mL DMSO was injected intothe nanoparticle solution. The mixture was shaken for 6 hours at 40° C.until it gradually turned heterogeneous, and precipitated down bycentrifugation (3,000 rpm, 10 minutes). The precipitate was carefullywashed with ethanol to remove excess DMSA, and dispersed in 10 mLethanol using a homogenizer. DMSA (50 mg) in 10 mL DMSO was added againto the nanoparticle-ethanol solution, and the whole process wasrepeated. The final precipitate was dispersed in 50 mL H₂O. TheDMSA-treated nanoparticles were eventually terminated with sulfhydryl(—SH) and unbound carboxylic acid; both chemical functional groups canbe used to conjugate specific targeting moieties. The number ofsulfhydryl groups per nanoparticle was about 50 as determined byEllman's reagent (Pierce Biotechnology). To conjugate DMSE-treatedFe@MnFe₂O₄ MNPs with (+)biotin-hydrazide (Aldrich), amide bonds wereformed on the Fe@MnFe₂O₄ MNPs using carboxylic acid and amine groupswere formed in biotin using NHS/EDC chemical reactions.

The DMSA-treated Fe@MnFe₂O₄ MNPs (25 mg) were dispersed in 10 mL H₂O,followed by the addition of NHS (3.5 mg), EDC (5 mg), and biotin (1 mg).The mixture was shaken for 3 hours at room temperature. The conjugatedFe@MnFe₂O₄ MNPs were then precipitated down (12,000 rpm, 20 minutes) andwashed three times with H₂O. To conjugate antibodies, the antibodieswere first rendered thiol active by attaching maleimide functionalgroups. The antibodies were then dissolved (anti-HER2/neu: Herceptin, 6mg/mL) in 1 mL PBS buffer and the pH was adjusted to 8.2 by adding 0.1 MNaHCO₃, and 1 mg sulfo-SMCC. The mixture was incubated for 30 minutes at37° C. The maleimide-active antibody was then purified with a PD-10desalting column (GE Healthcare Bio-Sciences) and immediately combinedwith the DMSA treated nanoparticles (5 mg/mL). The mixture was shakenfor 6 hours at 4° C., after which it was purified using Sephadex G-100(DNA grade, GE Healthcare Bio-Sciences). The number of antibodies pernanoparticle was about 10 as determined using bicinchoninic acid assays(BCA protein assay kit, Pierce Biotechnology).

Example 5 Surface Modification of MNPs and Cytotoxicity Test

To transfer as-synthesized, hydrophobic Fe@MnFe₂O₄ MNPs to aqueousbuffers, we modified the particle surface with 2,3-dimercaptosuccinicacid (DMSA) through ligand exchange. DMSA contains two carboxylic acid(—COOH) and two sulfhydryl (—SH) groups. When mixed with MNPs, thecarboxylic acid initially forms direct chelating bonds with theFe@MnFe₂O₄ MNP surface. FIG. 8 is a schematic diagram of Fe@MnFe₂O₄ MNPcoated with DMSA for conjugation with targeting moieties. The DMSAcoating is stabilized via intermolecular disulfide cross-linking amongDMSA molecules. Employing this method, we could reliably render theFe@MnFe₂O₄ MNPs water-soluble.

To conjugate targeting moieties to the particles, we utilized theterminal sulfhydryl or carboxylic acid functional groups. For instance,we could couple biotin molecules to the carboxylic acid groups of DMSAusing NHS/EDC chemistry (Nhydroxysuccinimide, NHS;N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide, EDC). Antibodies,separately modified with a maleimide functional group, were conjugatedto the sulfhydryl groups via disulfide bonding (Section VIII). Wesubsequently tested for potential cytotoxicity associated with theDMSA-coated Fe@MnFe₂O₄ MNPs. Normal (3T3 fibroblast) and cancer (HCT116) cells were cultured for 24 hours, following introduction ofdifferent quantities of MNPs. The cellular viability was then analyzedwith a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT)bromide assay (see Section VII). No acute cytotoxic effects wereobserved, which confirmed the biocompatibility of both the DMSA-coatedand the magnetic materials.

Example 6 Demonstration of Biological Applications Including AvidinTitration and Cellular Assay

To demonstrate the biological applications of Fe@MFe₂O₄ MNPs, we usedthe particles to sense biological markers with the NMR-based diagnosticplatform (DMR, diagnostic magnetic resonance). DMR sensing is based onthe change of transverse relaxation time (ΔT2) when detection targets insamples are recognized by MNPs. For molecular targets (e.g. proteins),the DMR assay employs the phenomenon of magnetic relaxation switching,in which MNPs are cross-linked with target molecules to formnanometer-scale clusters. For larger objects (e.g., bacteria, mammaliancells), targets are labeled with MNPs, and unbound particles are removedprior to measurements. For both assay configurations, the detectionsensitivity is commensurate with the r2 of MNPs.

Initially, we evaluated the sensing capacity of Fe@MFe₂O₄ MNPs inmolecular detection, using biotin-avidin interaction as a model.Titration experiments with biotinylated MNPs showed avidindose-dependent T2 changes. FIG. 9 shows a graph of normalized ΔT2 versusavidin concentration for several types of nanoparticles. Among the othertypes of MNPs, Fe@MnFe₂O₄ showed the highest sensitivity by detectingabout 1.5 pM of avidin; the DMR assay is as sensitive as ELISA but withthe added advantage of requiring much smaller samples (about 1 μl) andshorter assay times (less than 30 min).

Avidin titration: Avidin (ImmunoPure Avidin #21121; PierceBiotechnology) was dissolved in PBS. Samples were prepared by mixingvarious amounts of avidin to biotinylated Fe@MnFe₂O₄ MNPs. Following 15minutes of incubation at 37° C., the T2 values of samples were measuredon 1 μL samples using the miniaturized Nuclear Magnetic Resonance (NMR)system previously reported in “Chip-NMR biosensor for detection andmolecular analysis of cells,” (Lee et al., Nat. Med. 14, pp. 869-874(2008)). Carr-Purcell-Meiboom-Gill pulse sequences were used with thefollowing parameters: echo time (TE), 4 msec; repetition time (TR), 6sec; the number of 180° pulses per scan, 500; the number of scans, 8. Asa reference, ΔT2 was calculated using the T2 values of samples withoutavidin. All measurements were performed in triplicate and data weredisplayed as mean±standard error.

For cellular detection, we tagged cancer cells (SkBr3) with MNPsconjugated to antibodies that target a tumor marker (HER2). Followingthe MNP-incubation, the cellular relaxivities, defined as the relaxationrate (1/T2) per cell concentration, were found to be proportional to ther2 of the MNPs. FIG. 10A is a graph that shows relaxation rate per cellconcentration for several types of MNPs. Consequently, Fe@MnFe₂O₄produced the most pronounced ΔT2, which permitted target detection closeto the level of single cells. FIG. 10B is a graph that shows normalizedΔT2 versus cell concentration for Fe@MnFe₂O₄ MNPs.

Human SkBr3 breast cancer cells were cultured in vendor-recommendedmedia and maintained at 37° C. in a humidified atmosphere of 5% CO2 inair. At confluence, the cells were incubated with Fe@MnFe₂O₄ MNPsconjugated with HER2/neu antibodies for 10 minutes at 37° C. Referencesamples were prepared without targeting. All samples were triple-washedvia centrifugation (1,000 rpm, 5 minutes), resuspended in PBS, andserially diluted. The cell concentration in a sample was determinedusing a hemocytometer. The same miniaturized NMR system and the pulsesequences (described previously) were used to obtain T2 measurements.

Example 7 Magnetic Resonance Imaging (MRI)

Fe@MFe2O4 MNPs were evaluated for use as MRI imaging agents. Acomparative study of phantoms confirmed the use of Fe@MnFe2O4 forenhancing contrast. For example, compared to the widely-used CLIOnanoparticles, Fe@MnFe₂O₄ was able to produce the same signal changes atabout 10 times lower doses. We also performed preliminary in vivoimaging studies. We intravenously injected a variety of MNPs[cross-linked iron oxide (CLIO), Fe₃O₄, and Fe@MnFe₂O₄] into mice, whilekeeping the same metal dose (10 mg [metal]/kg). We then obtainedT2-weighted images at different time points using a 7 T MRI machine(Pharmascan, Bruker). Taken at 3 hours post-injection, the imagesverified that the contrast enhancement was indeed commensurate with ther2 value of each type of MNPs.

FIG. 11A is an MRI image of organs prior to the injection of MNPs. FIGS.11B to 11D are MRI images of organs in which different MNPs have beeninjected. The letter “L” in the images identifies the location of theliver whereas the letter “K” in the images identifies the location ofthe kidney. Fe@MnFe2O4 MNPs (FIG. 11B) resulted in the most significantdarkening, which supports the potential utility of Fe@MnFe2O4 MNPs as anefficient imaging agent. In organs containing phagocytic cells such asliver, spleen, or bone marrow, the peak signal changes occurred ataround 3 hours after intravenous administration, consistent with an hourlong blood half-life. At early time points, most organs includingkidneys showed considerable darkening due to vascular perfusion. Thesechanges all returned to baseline within 24 hours.

The imaging study was performed using a 4.7 T and a 7.0 T scanner(Bruker) and a volume coil in birdcage design (Rapid Biomedical,Germany). To determine r2, phantoms consisting of Fe@MnFe₂O₄ MNPs withdifferent metal concentrations were prepared and T2 values were measuredusing the following spin echo parameters: TE=10 msec; TR=2000 msec;matrix 256×256; number of 180° pulses per scan=16. For mouse imaging,Fe@MnFe₂O₄ MNPs (in PBS) were injected intravenously with a metal doseof 10 mg [metal]/kg via the tail vein. The animal was anesthetized withisoflurane (5% induction, 1.5% maintenance) in the mixed gas of N₂O andO₂ (7:3). T2-weighted images were then obtained using the followingparameters: TE=36 msec; TR=2420 msec; flip angle 90°; matrix 128×128;field of view 4×4 cm², slice thickness 1 mm.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A nanoparticle comprising: a solid ferromagneticcore; a super-paramagnetic shell on a surface of and surrounding themagnetic core, wherein the super-paramagnetic shell has a thicknessbetween about 0.5 nm and about 3 nm.
 2. The nanoparticle of claim 1,wherein the nanoparticle has a diameter greater than or equal to about 2nm.
 3. The nanoparticle of claim 1, wherein the ferromagnetic core has acore diameter in a range of about 1 nm to about 15 nm.
 4. Thenanoparticle of claim 1, wherein the ferromagnetic core comprises Fe,Co, Ni, FePt or SmCo.
 5. The nanoparticle of claim 1, wherein thesuper-paramagnetic shell comprises an oxide of a magnetic material. 6.The nanoparticle of claim 1, wherein the super-paramagnetic shellcomprises a dopant material.
 7. The nanoparticle of claim 6, wherein thedopant material comprises a metal selected from the group consisting ofMn, Co, Ni, Zn, and ZnMn.
 8. The nanoparticle of claim 1, furthercomprising a coating on the super-paramagnetic shell, wherein thecoating is configured to increase the aqueous solubility of thenanoparticle.
 9. The nanoparticle of claim 8, wherein the coatingcomprises 2,3-dimercaptosuccinic acid (DMSA).
 10. The nanoparticle ofclaim 1, further comprising a coating on the super-paramagnetic shell,wherein the coating is configured to bind the nanoparticle to a targetmolecule.
 11. The nanoparticle of claim 1, further comprising a dextranpolymer coating on the super-paramagnetic shell.